Airway epithelial NKCC1 shares properties with secretory forms of basolateral Na-K-2Cl cotransport by supplying Cl for secretion into the airway lumen. NKCC1 also has a non-secretory role in the maintenance of a periciliary fluid layer surrounding cilia, which are necessary for optimal mucociliary clearance. Compromising the periciliary fluid layer is thought to contribute to the pathophysiology of lung disorders, such as asthma, bronchitis, and cystic fibrosis. Our previous studies established a key role for protein kinase C-delta (PKC4) and its binding to the scaffold actin, in the activation of airway epithelial NKCC1 by multiple stimuli, including hyperosmotic stress, low intracellular Cl, and Gs coupled receptors. We identified a regulatory proteome, consisting of the N terminus of NKCC1, protein phosphatase 2A (PP2A), stress activated serine-threonine kinase SPAK and PKC4, but do not understand how the proteome regulates NKCC1 function. We also discovered the interaction of the C terminus (CT) of NKCC1 with a copper transport protein Murr1 (COMMD1) and loss of NKCC1 activation by hyperosmotic stress after downregulation of Murr1. We have yet to ascertain how Murr1 regulates NKCC1. The long term goal of our research is to understand how protein-protein interactions regulate NKCC1. We propose the following specific aims: 1) To test the hypothesis that PKC4 regulates the function of SPAK. We will correlate downregulation of SPAK, using silencing RNA, with activation of NKCC1 and of PKC4. We will investigate binding of PKC4 to SPAK and phosphorylation of SPAK by PKC4 and dephosphorylation by PP2A. Binding motifs for proteins interacting with SPAK will be determined and the thermodynamics of binding established by oxidative footprinting. 2) To test the hypothesis that Murr1 regulates NKCC1 surface expression. We will determine a) whether Murr1 is necessary for surface expression and function of NKCC1 by downregulating Murr1 using silencing RNA, b) the turnover time of NKCC1 in the basolateral membrane and its regulation by actin and Murr1 and c) binding motifs for Murr1 and CT-NKCC1, using novel methods of oxidative footprinting and mass spectrometry, from which we will develop inhibitory peptides to alter end responses, including protein interaction, NKCC1 activity, and NKCC1 surface expression. 3) To test the hypothesis that phosphorylation of NKCC1 modulates its activity. We will determine a role for SPAK and PP2A in NKCC1 phosphorylation. We plan to test peptides encoding putative phosphorylation sites for inhibition of NKCC1 activation and plan to downregulate the critical enzyme and correlate loss of mass and activity with effects on NKCC1 function. Project Narrative: This project is an important first step in understanding the importance and prominent role of protein interactions in the regulation of Na-K-2Cl activity. New information gained from these studies will take us one step further toward correcting defective NKCC1 function and lead to the development of unique therapeutic tools to promote NKCC1 function in disease states. More importantly, the studies will lay the foundation for predicting and testing the effects of drugs, both pharmaceutical and recreational, that alter NKCC1 function.